The Nitrate Elimination Co., Inc. (NECi), an environmental biotechnology company, develops and commercializes enzyme-based products for testing and remediation of compounds of environmental concern. Current company focus is on the application of purified and stabilized higher plant Nitrate Reductase (NaR) for use in nitrate test kits, nitrate biosensors, and in immobilized enzyme nitrate elimination water- treatment technology. Contamination of water by excess nitrate is a growing pollution and human health problem worldwide. We propose to produce recombinant NaR in order to make NaR-based products more affordable and cost-competitive. Our working hypothesis is that recombinant, catalytically active NaR can be produced in the yeast Pichia pastoris, using existing squash and corn cDNA clones which express epitopes for NECi proprietary monoclonal antibodies. Recent data from NECi senior scientist's academic research program show that active NaR can be produced in and isolated from Pichia. Recombinant NaR will be purified using existing immunoaffinity chromatographic methods or alternative affinity methods. It will be performance tested in NECi prototype products by comparison with natural NaR. Fermentation parameters an optimal gene construction for maximal NaR production will be investigated in Phase II. NECi will develop core technology for production of complex recombinant enzymes with unique, value-added applications. PROPOSED COMMERCIAL APPLICATION: Consumption of excess nitrate causes health problems in humans, including methemoglobinemia in infants. Products for detection and remediation of nitrate have numerous markets and applications in testing and monitoring of foods and water, and in water treatment. NECi has established NaR as a safe and environmentally benign commercial reagent for nitrate testing. Recombinant NaR will improve competitiveness of enzyme-based products. In Phase II, application of CLECs (Cross-Linked Enzyme Crystal) technology to NaR will further enhance NaR stability and market value.